Methods for predicting the risk of diabetic nephropathy using genetic markers and arrays containing the same

ABSTRACT

A method for detecting a Chinese diabetic subject suffering from, at risk for developing, or suspected of suffering from a nephropathy. The method includes determining whether a sample from the subject has at least one of the following polymorphic sequences: an I/D genotype of an ACE gene, an M235T genotype of an AGT gene, a (CA)n-5′(z−2) genotype of an ALR2 gene, an C106T genotype of an ALR2 gene in the promoter region, a G-308A genotype of a TNF-α gene, or a complement thereof, provided that the ALR2 gene cannot be used alone, in which the presence of the polymorphic sequence indicates the subject suffering from, at risk for suffering from a nephropathy. An array for detecting a Chinese diabetic subject suffering from, or at risk for suffering from, a nephropathy.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/591,824, filed Sep. 6, 2006, which is the U.S. National Phase filingunder 35 U.S.C.§371 of PCT/CN2005/000508, filed Apr. 15, 2005, whichdesignated the United States and was published in English, which claimspriority under 35 U.S.C. §119(a)-(d) to Chinese Patent Application No.200410033864.X, filed Apr. 15, 2004. The content of these applicationsis incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a method and an array for detecting asubject of Chinese descent suffering from, at risk for developing, orsuspected of suffering from a nephropathy, by using one or more asmarkers, and particularly to a method and an array for detecting anephropathy using one or more genetic polymorphisms selected from genesACE, AGT, ALR2, and TNF-α, provided that ALR2 gene cannot be used alone.

BACKGROUND OF THE INVENTION

Diabetic nephropathy is a leading cause of morbidity and mortality indiabetic patients. With the rising epidemic of diabetes in bothdeveloping and developed countries, diabetes is now the leading cause ofend stage renal disease (ESRD), accounting for 40-50% of all newpatients on renal replacement therapy (Ritz E, Rychlik I, Locatelli F,Halimi S, End-stage renal failure in type 2 diabetes: a medicalcatastrophe of worldwide dimensions, Am J Kidney Dis, 1999; 34:795-808).China is one of the 3 top countries with the most number of diabeticpeople estimated to increase to 40 million in 2025 with the predominantincrease occurring in the middle aged population (Chan J. C. N, Ng M. C.Y, Critchley J. A. J. H, Lee S. C, Cockram C. S, Diabetes mellitus—aspecial medical challenge from a Chinese perspective, Diabetes Researchand Clinical Practice, 2001; 54:S19-27). This is mainly due to therising prevalence of young onset diabetes and childhood obesity andmetabolic syndrome (Chan J. C. N, Ng M. C. Y, Lessons learned from youngonset diabetes in China, Current Diabetes Report, 2003; 3:101-107; ChanJ. C. N, Cheung C. K, Cheung M. Y. F, Swaminathan R, Critchley J. A. J.H, Cockram C. S, Abnormal albuminuria as a predictor of mortality andrenal impairment in Chinese patients with NIDDM, Diabetes Care, 1995;18:1013-1014; Chan J. C. N, Ko G. T. C, Leung D, Cheung R. C. K, CheungM, So W. Y, et al, The long term effects of angiotensin convertingenzyme inhibition and metabolic control on cardiovascular and renaloutcomes in hypertensive Type 2 diabetic patients, Kidney International,2000; 57:590-600). In the World Health Organization Multinational Studyfor Vascular Diseases in Diabetes (WHO-MSVDD), Asian, notably Chineseand Japanese patients, had higher incidence of ESRD than Caucasian type2 diabetic patients (Morrish N. J, Wang S, Stevens L. K, Fuller J. H,Keen H, Mortality and causes of death in the WHO Multinational Survey ofVascular Diseases in Diabetes, Diabetologia, 2001; 44:S14-21).

In contrast to the Caucasian diabetic population in whom the majority ofpatients die from cardiovascular events, ESRD is an important cause ofdeath in Chinese diabetic patients (Chan J. C. N, Cheung C. K, Cheung MY F, Swaminathan R, Critchley J. A. J. H, Cockram C. S, Abnormalalbuminuria as a predictor of mortality and renal impairment in Chinesepatients with NIDDM, Diabetes Care, 1995; 18:1013-1014). These findingshave recently been confirmed by the WHO-MSVDD (Morrish N. J, Wang S,Stevens L. K, Fuller J. H, Keen H, Mortality and causes of death in theWHO Multinational Survey of Vascular Diseases in Diabetes, Diabetologia,2001; 44:S14-21). In keeping with these findings, it is now recognizedthere are inter-ethnic differences in the allele frequency or haplotypesof disease-associated genes which may contribute to the racialdifferences in susceptibility to disease development (Ng M, Wang Y, SoW, Cheng S, Visvikis S, Zee R, et al, Ethnic differences in the linkagedisequilibrium and distribution of single nucleotide polymorphisms in 35candidate genes for cardiovascular diseases, Genomics, 2003: in press;Young R P, Thomas G N, Critchley J. A. J. H, Tomlinson B, Woo K. S,Sanderson J. E, Interethnic differences in coronary heart diseasemortality in 25 populations: associations with the angiotensinconverting enzyme DD genotype frequency, Journal of Cardiovascular Risk,1998; 5:303-7). Given the effect of duration of disease on developmentof complications, rising prevalence of young and middle aged diabeticpopulation and the racial predilection to develop diabetic renaldisease, there is a looming epidemic of renal failure and cardiovasculardiseases in our increasingly young population with its socioeconomicimplications (Chan J. C. N, Ng M. C. Y, Critchley J. A. J. H, Lee S. C,Cockram C. S, Diabetes mellitus—a special medical challenge from aChinese perspective, Diabetes Research and Clinical Practice, 2001;54:S19-27).

We have previously reported the high prevalence of nephropathy rangingfrom 30% to 50% and the predictive value of this urinary marker formortality and deterioration of renal function in Chinese diabeticsubjects (Chan J C N, Cheung C K, Cheung M Y F, Swaminathan R, CritchleyJ A J H, Cockram C S, Abnormal albuminuria as a predictor of mortalityand renal impairment in Chinese patients with NIDDM, Diabetes Care,1995; 18:1013-1014; So W Y, Chan N, Tong P C Y, Chow C C, Chan W B, Ng MC Y, Chan J C N, Effect of RAAS inhibition on survival and renaloutcomes in 3737 Chinese Type 2 diabetic patients, Hypertension. 2004;44: 294-9).

Apart from hypertension and dyslipidemia, we have reported theindependent association between insulin resistance and diabeticnephropathy (Chan J. C. N, Tomlinson B, Nicholls M. Q Swaminathan R,Cheung C. K, Cockram C S, Albuminuria, insulin resistance anddyslipidaemia in Chinese patients with non-insulin-dependent diabetes(NIDDM), Diabetic Medicine, 1996; 13:150-55) as well as the intimaterelationships between obesity, albuminuria and dysglycemia in Chinesesubjects (Lee Z, Critchley J, Ko G T, Anderson P, Thomas N, Young R, etal, Obesity and cardiovascular risk factors in Hong Kong Chinese,Obesity Reviews, 2002; 3:178-182). Family-based studies and segregationanalysis (The Diabetes Control and Complications Trial Research Group,Clustering of long term complications in families with diabetes in thediabetes control and complication trial, Diabetes, 1997; 46:1829-1839)as well as genome scan (Imperatore G Hanson R L, Pettitt D, Kobes S,Bennett P, Knowler W, Sib pair linkage analysis for susceptibility genesfor microvascular complications among Pima Indians with type 2 diabetes.Pima Diabetes Gene Group, Diabetes, 1998; 47:821-30; Imperatore QKnowler W, Pettitt D, Kobes S, Bennett P, Hanson R, Segregation analysisof diabetic nephropathy in Pima Indians, Diabetes, 2000; 49:1049-56)have confirmed strong genetic components in the development of diabeticrenal disease. The renin angiotensin system (RAS) plays a pivotal rolein the regulation of systemic and renal haemodynamics as well ascellular and tissue growth (Cooper M, Pathogenesis, prevention andtreatment of diabetic nephropathy, Lancet, 1998; 352:213-9). The TTgenotype of the AGT M235T polymorphism and the D allele of the ACE I/Dpolymorphism have been associated with diabetic nephropathy inCaucasian, Japanese and Chinese diabetic patients (Fujisawa T, IkegamiH, Kawaguchi Y, Hamada Y, Ueda H, Shintani M, et al, Meta analysis ofassociation of insertion/deletion polymorphism of angiotensin Iconverting enzyme gene with diabetic nephropathy and retinopathy,Diabetologia, 1998; 41:47-53; Young R. P, Chan J. C. N, Poon E,Critchley J. A. J. H, Cockram C. S, Associations between albuminuria andangiotensinogen T235 and angiotensin converting enzymeinsertion/deletion polymorphisms in Chinese NIDDM patients, DiabetesCare, 1997; 21:431-7; Ringel J, Beige J, Kunz R, Distler A, Sharma A,Genetic variants of the renin angiotensin system, diabetic nephropathyand hypertension, Diabetologia, 1997; 40:193-9, Wang Y, Ng M, So W, TongP, Ma R, Chow C, Cockram C, J C N Chan. Prognostic effect ofinsertion/deletion polymorphism of the ace gene on renal andcardiovascular clinical outcomes in Chinese patients with type 2diabetes. Diabetes Care, 2004; 28: 348-54).

Tumor necrosis factor alpha (TNF-α), a cytokine secreted by adipocytes,is the linking factor for obesity-related insulin resistance(Hotamisligil G S, Spiegelman B M, Tumor necrosis factor: a keycomponent of the obesity-diabetes link, Diabetes, 1994; 43:1271-8), thelatter being an important feature of diabetic nephropathy, includingChinese patients (Chan J. C. N, Tomlinson B, Nicholls M. G S SwaminathanR, Cheung C. K, Cockram C. S, Albuminuria, insulin resistance anddyslipidaemia in Chinese patients with non-insulin-dependent diabetes(NIDDM), Diabetic Medicine, 1996; 13:150-55). A recent Japanese studyhas shown a positive association between elevated serum TNF-α level andnephropathy in Type 2 diabetic patients (Moriwaki Y, Yamamoto T,Shibutani Y, Aoki E, Tsutsumi Z, Takahashi S, et al, Elevated levels ofinterleukin 18 and tumor necrosis factor alpha in serum of patients withtype 2 diabetes mellitus: relationship with diabetic nephropathy,Metabolism: Clinical and Experimental, 2003; 52:605-8). In this regard,the G-308A polymorphism in the promoter region of TNF-α gene had beenreported to be associated with obesity and insulin resistance (DalzielB, Goskby A, Richman R, Bryson J, Caterson I, Association of TNF alpha-308 G/A promoter polymorphism with insulin resistance in obesity,Obesity Research, 2002; 10:401-7) and increased transcriptional activityof TNF-α (Kroeger K, Carville K, Abraham L, The -308 tumor necrosisfactor alpha promoter polymorphism effects transcription, MolecularImmunology, 1997; 34:391-99). Aldose reductase (ALR2) is the key enzymein the polyol pathway which can lead to increased oxidative stress andalteration of intracellular milieu causing diabetic microangiopathy(Hodgkinson A, Sondergaard K, Yang B, Cross D, Millward B, Demaine A,Aldose reductase expression is induced by hyperglycemia in diabeticnephropathy, Kidney International, 2001; 60:211-8). Both the z−2 alleleof 5′-(CA) and T allele of C-106T polymorphisms of this gene have beenshown to increase risk for nephropathy in Type 1 and Type 2 diabeticpatients including Chinese (Wang Y, Ng M, Lee S, So W, Tong C, CockramC, et al, Phenotypic heterogeneity associations of two aldose reductasegene polymorphisms with nephropathy and retinopathy in Type 2 diabetes,Diabetes Care, 2003; 26:2410-5). These putative genetic factors furtherinteract with metabolic, hemodynamic and growth factors to causeproteinuria and progressive decline in renal function (Parving H. H,Tarnow L, Rossing P, Genetics of diabetic nephropathy, Journal ofAmerican Society of Nephrology, 1996; 7:2509-17).

Although there have been reports on the associations between these 5genetic markers and diabetic complications in Caucasian and Japanesepopulations, there have been scanty and inconsistent reports in Chinesediabetic populations. To date, there have been no reports showing theinteractive effects of these genetic factors on development of diabeticcomplications including diabetic nephropathy.

One of the promises of applied genomics lies in its potential use toidentify at risk subjects for early and targeted intervention topreserve health and reduce the impact of killing diseases such asdiabetes (Collins F, Green E, Guttmacher A, Guyer MobotuNHGRI, A visionfor the future of genomics research. A blueprint for the genomic era,Nature, 2003; 422:835-47). In our search for genetic factors to identifyhigh risk subjects for complications in Chinese diabetic patients, wewere the first group to report the association between the AGT TTgenotype and diabetic nephropathy and its synergistic effects with ACE Dallele on its development (Young R P, Chan J. C. N, Poon E, Critchley J.A. J. H, Cockram C. S, Associations between albuminuria andangiotensinogen T235 and angiotensin converting enzymeinsertion/deletion polymorphisms in Chinese NIDDM patients, DiabetesCare, 1997; 21:431-7) as well as the independent predictive role of ACEDD genotype on development of ESRD (Wang Y, Ng M, So W, Tong P, Ma R,Chow C, Cockram C, JCN Chan. Prognostic effect of insertion/deletionpolymorphism of the ace gene on renal and cardiovascular clinicaloutcomes in Chinese patients with type 2 diabetes. Diabetes Care 2004;28:348-54). Similarly, we were also the first group to report the riskassociation between ALR2 TT genotype (Wang Y, Ng M, Lee S, So W, Tong C,Cockram C, et al, Phenotypic heterogeneity associations of two aldosereductase gene polymorphisms with nephropathy and retinopathy in Type 2diabetes, Diabetes Care, 2003; 26:2410-5) and diabetic nephropathy insubjects of Chinese descent.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a method for detecting aChinese diabetic subject suffering from, at risk for developing, orsuspected of suffering from a nephropathy, the method comprising thestep of:

determining whether a sample from the subject has at least onepolymorphic sequence selected from the group consisting of polymorphicsequences an I/D genotype of an ACE gene, an M235T genotype of an AGTgene, a (CA)n-5′(z−2) genotype of an ALR2 gene, a C106T genotype of anALR2 gene in the promoter region, a G-308A genotype of a TNF-α gene, anda complement thereof, provided that the ALR2 gene cannot be used alone,wherein the presence of the polymorphic sequence indicates the subjectsuffering from, or at risk for suffering from the nephropathy.

In an embodiment of the invention, the method may further comprise thestep of providing a sample from the subject. The subject is preferablysuffering from Type 2 diabetes. The sample is preferably blood.

The present invention also pertains to an array for detecting a subjectof Chinese descent suffering from, at risk for developing, or suspectedof suffering from a nephropathy, comprising at least one polymorphicsequence selected from the group consisting of sequences: an I/Dgenotype of an ACE gene, an M235T genotype of an AGT gene, a(CA)n-5′(z−2) genotype of an ALR2 gene, an C106T genotype of an ALR2gene in the promoter region, a G-308A genotype of a TNF-α gene, and acomplement thereof.

In the invention, the I/D polymorphism preferably comprises a DDgenotype, and the G-308A polymorphism preferably comprises a GG genotype

The present invention still relates to a kit for detecting a subject ofChinese descent suffering from, at risk for developing, or suspected ofsuffering from a nephropathy. The kit generally comprises an arraydefine herein, and an instructional material teaching the processing ofthe sample with the array. Preferably, the kit may comprise a device forobtaining a sample from a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the odds ratio of having nephropathy in the 711 ChineseType 2 diabetic patients with different number of risk genotypesincluding a TT genotype of an AGT gene, a DD/DI genotype of an ACE gene,a GG genotype of an TNF-α gene, an x/z−2 or z−2/z−2 genotype and a CT/TTgenotype of an ALR2 gene.

FIG. 2 shows Kaplan-Meier curves for renal endpoint among carriers ofACE gene I/D genotypes in 947 Chinese Type 2 diabetic patients.

FIG. 3 shows Kaplan-Meier curves for the primary composite endpoint ofend-stage renal disease or all-cause death (Panel A), end-stage renaldisease (Panel B), and all-cause death (Panel C) in patients managed bya multidisciplinary team according to a protocol with particularemphasis on periodic monitoring, patient compliance and treatment totarget (intervention group) versus usual clinic-based care (controlgroup) where adherence to treatment guideline by doctors and patientcompliance remained uncertain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Genetic, epidemiological and experimental studies support the notionthat multiple biochemical pathways are involved in diabetic nephropathy(Cooper M, Pathogenesis, prevention and treatment of diabeticnephropathy, Lancet, 1998; 352:213-9). Based on international and localas well as clinical and experimental evidence, we considered the TTgenotype of AGT gene M235T, DD/ID genotype of ACE gene I/D, GG genotypeof TNF-α gene G-308A, x/z−2 or z−2/z−2 genotype (x=any allele ratherthan z−2) and CT/TT genotype of ALR2 gene as potential risk genotypesfor diabetic nephropathy in Chinese subjects.

DEFINITIONS

Unless specified otherwise in the invention, the term “AGT gene M235T”or “AGT M235T” is meant to “a M235T genotype of an AGT gene”; the termof “ACE gene I/D” or “ACE I/D polymorphism” has the same meaning of “anI/D genotype of an ACE gene”; the term of “GG of TNF-α gene” isequivalent to “GG genotype of TNF-α gene G-308A”; the term of “TNF-αgene G-308A” or “G-308A polymorphism in the promoter region of TNF-αgene” or “TNF-α G-308A” or “TNF α G308A polymorphism” is meant to “aG-308A genotype of a TNF-α gene”; the term of “z−2 allele of 5′-(CA)n ofAldose reductase (ALR2)” or “ALR2 (CA)n-5′(z−2)” is equivalent to “a(z−2) genotype of an ALR2 gene 5′-(CA) repeats”; the term “T allele ofC-106T polymorphisms of Aldose reductase (ALR2)” is equivalent to “ALR2TT genotype, TT of ALR2 gene” or “CT/TT genotype of ALR2 gene”; and theterm of “a C-106T polymorphism of ALR2” is meant to “a C106T genotype ofan ALR2 gene in the promoter region”.

In the method in accordance with present invention for detecting aChinese diabetic subject suffering from, at risk for developing, orsuspected of suffering from a nephropathy, which comprises the step ofdetermining whether the sample has at least one polymorphic sequenceselected from the group consisting of polymorphic sequences an I/Dgenotype of an ACE gene, an M235T genotype of an AGT gene, a(CA)n-5′(z−2) genotype of an ALR2 gene, an C106T genotype of an ALR2gene in the promoter region, a G-308A genotype of a TNF-α gene, and acomplement thereof, provided that the ALR2 gene cannot be used alone,wherein the presence of the polymorphic sequence indicates the subjectsuffering from, at risk for, or suspected of suffering from kidneydiseases.

Polymorphisms as genetic markers used in the method can be identified asfollows:

(a) extracting a genomic DNA from a subject;

(b) amplifying by a PCR the genomic DNAs of ACE gene, AGT, TNF-α G-308Apolymorphism, aldose reductase (ALR2) CA repeat, and promoter C106T ofthe ALR2 gene with the template of the human genomic DNA; and

(c) identifying the product of step (b) by gel-separation or sequencing.

In the present invention, the genomic DNA may be extracted from a fluidof the subject such as blood and urine. Blood is preferable.

Although the method for detection of the invention can be used for asubject, it is preferred for a subject who is suffering from Type 2diabetes. The subject of Chinese descent is particularly suitable forthe invention.

The four candidate genes selected in this study can all contribute tothe development of diabetic nephropathy through plausible metabolicpathways. The AGT and ACE are important components of the RAS whichcontribute to hypertension and abnormal tissue growth (Cooper M,Pathogenesis, prevention and treatment of diabetic nephropathy, Lancet,1998; 352:213-9; Ringel J, Beige J, Kunz R, Distler A, Sharma A, Geneticvariants of the renin angiotensin system, diabetic nephropathy andhypertension, Diabetologia, 1997; 40:193-9). The cytokine TNF-αrepresents a link between obesity and insulin resistance (Hotamisligil GS, Spiegelman B M, Tumor necrosis factor: a key component of theobesity-diabetes link, Diabetes, 1994; 43:1271-8) and its serum levelhas been reported to be positively correlated with diabetic nephropathy(Moriwaki Y, Yamamoto T, Shibutani Y, Aoki E, Tsutsumi Z, Takahashi S,et al, Elevated levels of interleukin 18 and tumor necrosis factor alphain serum of patients with type 2 diabetes mellitus: relationship withdiabetic nephropathy, Metabolism: Clinical and Experimental, 2003;52:605-8). The ALR2 contributes to development of diabeticmicroangiopathy through intracellular accumulation of sorbitol andincreased oxidative stress under hyperglycemic conditions (Chung S, HoE, Lam K, Chung S, Contribution of polyol pathway to diabetes-inducedoxidative stress, Journal of American Society of Nephrology, 2003;14:S233-6).

These genotypic findings in Chinese populations are based on a series ofcross-sectional, prospective and case-control studies and are inconsistence with the phenotypic features of patients with diabeticnephropathy who were more obese, hypertensive and had more adverse lipidand glycemic control than patients without nephropathy. Large scalerandomized clinical studies have also confirmed the beneficial effectsof inhibition of the RAS as well as improved glycemic and blood pressurecontrol on diabetic proteinuria and development of ESRD (Brenner B. M,Cooper M. E, De Zeeuw D, Keane W. F, Mitch W. E, Parving H. H, et al,Effects of Losartan on renal and cardiovascular outcomes in patientswith type 2 diabetes and nephropathy, New England Journal of Medicine,2001; 345:861-9; UKPDS, Intensive blood glucose control withsulphonylureas or insulin compared with conventional treatment and riskof complications in patients with type 2 diabetes (UKPDS 33), Lancet,1998; 352:837-53; Adler A. I, Stratton I. M, Neil H. A, Yudkin J. S,Matthews D. R, Cull C. A, et al, Association of systolic blood pressurewith macrovascular and microvascular complications of type 2 diabetes(UKPDS 36): prospective observational study, British Medical Journal,2000; 321:412-9). More recently, combination therapy with ACE and ALR2inhibitors have been shown to act synergistically to improve nervefunction in diabetic rats (Cotter M, Mirrlees D, Cameron N,Neurovascular interactions between aldose reductase and angiotensinconverting enzyme inhibition in diabetic rats, European Journal ofPharmacology, 2001; 417:223-30). Taken together, identification of thesegenetic factors and their interactions (and the associated microchiptechnology to increase the efficiency of simultaneous screening forthese risk genotypes in the same subject) should allow selection of highrisk individuals for intensified and targeted therapy to reduce risk ofcomplications. In this regard, we have demonstrated that a multifacetedapproach using a multidisciplinary team with particular focus onperiodic monitoring, treatment to target and reinforcement of patientcompliance was associated with 40-70% risk reduction in development ofdeath and ESRD in Chinese diabetic patients (FIG. 3). In light of thefurther increase in risk profile of subjects carrying these genotype(s),the benefits of this disease management protocol and itscost-effectiveness should be further enhanced.

After adjustment for age and sex, we found that the risk for havingnephropathy increased progressively and significantly with increasingnumber of risk genotypes. Patients with 3 or more risk genotypes whoaccounted for 66% of our subjects, had 1.8-2.0 fold increased risk fordiabetic nephropathy, compared to those patients with 0 or 1 riskgenotype.

We have produced original data to confirm the predictive roles of DDgenotype on development of ESRD, that of TNF-α on diabetic nephropathyespecially in obese diabetic patients and more importantly, theinteractions between these 5 genotypes on development of diabeticnephropathy.

It is understood that one or more genotypes mentioned above can be usedto develop arrays that are used in conjunction with other knownclinical, biochemical and genetic for predicting the risk of diabeticcomplications including nephropathy in Chinese diabetic patients, andthese genotypes or equivalent arrays thereof can be used to identify atrisk subjects for diabetes and/or diabetic renal disease for riskmodification using a multifaceted approach including intensivemonitoring, pharmacological and non-pharmacological therapy.

To practice the invention, it is convenient to use an array definedherein as a kit for detecting a Chinese diabetic subject suffering from,at risk for developing, or suspected of suffering from kidney diseases.The kit includes the array and a pair of primers for amplifying thegenes ACE, AGT, ALR2 or TNF-α. In an embodiment of the kit of theinvention, the primers are selected from SEQ ID NO: 1 to SEQ ID NO: 10.

The present invention also provides a kit which includes an arraycomprising at least one polymorphic sequence selected from the groupconsisting of: an I/D genotype of an ACE gene, an M235T genotype of anAGT gene, a (CA)n-5′(z−2) genotype of an ALR2 gene, an C106T genotype ofan ALR2 gene in the promoter region, a G-308A genotype of a TNF-α gene,and a complement thereof, and optionally probes designed by thesequences as controls.

The present invention will be further described with the followingExamples.

EXAMPLE 1 Identification of the Genotype of Five Polymorphic SequencesPreparation of Human Genomic DNA

About 10 ml of EDTA blood was collected from each subject. The genomicDNA was extracted by lysing the cells with SDS and proteinase Kovernight followed by extracting with phenol and chloroform. The DNApellet was then dissolved in 1×TE buffer. The quantity and quality ofthe extracted DNA were determined by taking the optical densities at 260nm and 280 nm. The extracted DNA was stored at 4° C. for next genotypingassays.

PCR Conditions for ACE Genes

Reactions were performed according to Rigat's Method (Rigat B, Hubert C,Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F, An insertion deletionpolymorphism in angiotensin I converting enzyme gene accounting for halfthe variance of serum enzyme levels, Journal of Clinical Investigation,1990; 86:1343-1346) with modifications. 150 ng DNA templates wereamplified using GeneAmp PCR System 9700 (ABI) in standard PCR buffer (50mM KCl, 10 mM Tris-HCl, pH 8.3, 3 mM MgCl₂, 0.2 mM each dNTP) (ABI) witha primer concentration of 5 μmol each and 0.6 U Tag Polymerase(Amersham). Total volume was 20 μl. The cycling conditions were asfollows: initial denaturation at 94° C. for 2 min., 94° C. for 1 min.,58° C. for 1 min., 72° C. for 2 min. for a total of 30 cycles, and finalextension 72° C. for 5 min. The sequence of the primer was:

SEQ ID NO. 1 5′CTG GAG ACC ACT CCC ATC CTT TCT 3′ SEQ ID NO. 2 5′GAT GTGGCC ATC ACA TTC GTC AGA T 3′

The PCR product was a 190 by fragment for the deletion and a 490 byfragment for the presence of insertion allele.

PCR Conditions for Angiotensinogen Genes

Reactions were performed according to Russ's Method (Russ A, Maerz W,Ruzicka V, Stein U, Gross W, Rapid detection of the hypertensionassociated Met235→Thr allele of the human angiotensinogen gene, HumanMolecular Genetics, 1994; 2:609-10). The PCR was performed in standardbuffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl₂, 50 uM eachdNTP) (ABI) with a primer concentration of 0.3 uM each and 0.75 U TagPolymerase (Amersham) with 200 ng DNA template in GeneAmp PCR system9700 (ABI). Total volume was 20 μl. The sequence of the primer used inthe reaction was:

SEQ ID NO. 3 5′-CAG GGT GCT GTC CAC ACT GGA CCC C-3′ SEQ ID NO. 4 5′-CCGTTT GTG CAG GGC CTG GCT CTC T-3′Cycling conditions were as follows: denaturation at 90° C. 3 min., 10cycles of 94° C. 1 min., 68° C. 1 min., 72° C. 1 min., followed by 30cycles 90° C. 30 sec., 68° C. 1 min., and 72° C. 30 sec., a finalextension 72° C. 10 min.

The PCR products were digested with 5 U Tth 111 I (Promega) at 65° C.overnight. The digested fragments were separated by electrophoresis in3% agarose gel. The homozygous methionine allele appeared as anon-digested single 165 bp and threonine allele as digested 141 and 24bands.

PCR Conditions for TNF-α G-308A Polymorphism

Reactions were performed as described by Wilson et al (Wilson A, diGiovine F, Blakemore A, Duff G, Single base polymorphism in the humantumor necrosis factor alpha gene detectable by NcoI restriction of PCRproduct, Human Molecular Genetics, 1992; 1:535). The reaction wasperformed with GeneAmp PCR system 9700 (ABI) in a final volume of 20 μlcontaining 100 ng DNA template, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5mM MgCl₂, 0.2 mM each dNTP (Boehringer-Mannheim, Germany) using a primerconcentration of 0.5 mM each and 0.05 U Taq polymerase(Boehringer-Mannheim, Germany). The sequence of the primer used in thereaction was as follows:

5′-AGG CAA TAG GTT TTG AGG GCC AT-3′ SEQ ID NO. 5 5′-TCC TCC CTG CTG CTCCGA TTC CG-3′ SEQ ID NO. 6

Cycling conditions were as follows: initial denaturation at 95° C. for 3min., 35 cycles of 95° C. 1 min., 58° C. 1 min., and 72° C. 1 min.,final extension 72° C. 10 min. The amplified PCR products were digestedwith 10 U NcoI (Promega) at 37° C. overnight. The digested fragmentswere separated by electrophoresis in 3% agarose gel. The A allele gave aband size of 107 bp while the G allele produced 87 bp and 20 bpfragments.

PCR Conditions for Aldose Reductase (ALR2) CA Repeats

The region containing CA dinucleotide repeat was amplified by PCR withprimers that flanked a 138 by region using the method described by Ko etal (Ko B. C. B, Lam K. S. L, Wat N. M. S, Chung S. S. M, An (A-C)ndinucleotide repeat polymorphic marker at the 5′ end of the aldosereductase gene is associated with early onset diabetic retinopathy inNIDDM patients, Diabetes, 1995; 44:727-32). The forward primer used inthe reaction was as follows:

5′-GAA TCT TAA CAT GCT CTG AAC C-3′ SEQ ID NO. 7

and the reverse primer was:

SEQ ID NO. 8 Arpr2 5′-GCC CAG CCC TAT ACC TAG T-3′.

An M13 tail (5′-CAC GAC GTT GTA AAA CGA C-3′) was added to 5′ end of theforward primer for labeling of infrared fluorescence.

A PCR was carried out in a total volume of 4 μl with 1 ng genomic DNA,2.5 mM MgCl₂, 0.2 mM of each dNTP, 0.1 pmol/ul of each primer, 0.15pmol/ul of IRD800 labeled M13 forward (−29) primer, and 0.15 U Taqpolymerase (Amplitaq, Perkin-Elmer/Cetus, Norwalk, Conn.) in the bufferprepared according to the supplied recipe. Cycling conditions were asfollows: initial denaturation at 94° C. for 3 min., 35 cycles 94° C. 1min., 57° C. 1 min., 72° C. 1 min., and a final extension 72° C. 10 min.

The amplified PCR products were heated at 95° C. for 5 mins and thenloaded onto 5.5% denaturing polyacrylamide gel and resolvedelectrophoretically in 0.8×TBE at constant power 75 W and 55° C. usingLi-COR DNA Analyser (Li-COR, Lincoln, Nebr.). Alleles were sized bycomparing a plasmid DNA containing a 23(CA) repeats of ALR2 gene whichwas kindly provided by Dr. Shiro Maeda from Shiga University of MedicalScience of Japan.

PCR Conditions for Promoter C106T of ALR2 Genes

Reactions were performed as described by Kao Y L et al (Kao Y, DonaghueK, Chan A, Knight J, Silink M, A novel polymorphism in the aldosereductase gene promoter region is strongly associated with diabeticretinopathy in adolescents with type 1 diabetes, Diabetes, 1999;48:1338-40). The reaction was performed with GeneAmp PCR system 9700(ABI) in a final volume of 20 ul containing 100 ng DNA template, 50 mMKCl, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl₂, 0.2 mM each dNTP(Boehringer-Mannheim, Germany) using a primer concentration of 0.5pmol/μl each and 0.5 U Taq polymerase (Boehringer-Mannheim, Germany).The sequence of the primer used was as follows:

5′-CCT TTC TGC CAC GCG GGG CGC GGG-3′ SEQ ID NO. 9 5′-CAT GGC TGC TGCGCT CCC CAG-3′ SEQ ID NO. 10Cycling conditions were as follows: initial denaturation at 94° C. for 3min., 35 cycles of 94° C. 1 min., 57° C. 1 min., and 72° C. 1 min., afinal extension 72° C. 10 min. The amplified PCR products were digestedwith 5 U BfaI (New England Biolabs, Beverly, Mass.) at 37° C. overnight.The digested fragments were separated by electrophoresis in 3.5% agarosegel. The C allele was indicated by 206 bp and 57 bp fragments while the206 bp fragment was further cleaved into 147 bp and 59 bp for the Tallele.

EXAMPLE 2 Relationship of Gene-Gene Interactions with Nephropathy

We examined the interactive effects of AGT gene M235T, ACE (I/D), TNF-αgene G-308A, ALR2 gene 5′-(CA)_(n) and promoter C-106T polymorphisms in711 Chinese Type 2 diabetic patients (303 male and 408 female, aged63.1±11.1 years). Patients who had duration of diabetes>10 years andplasma creatinine<100 μmol/l and spot urine albumin creatinine ratio(ACR)<3.5 mg/mmol were selected as control cases (n=388). Patients whohad either plasma creatinine ≧150 μl mol/l or ACR ≧25 mg/mmol wereconsidered to have nephropathy (n=323). Statistical Package for SocialScience (Version 10.0, SPSS Inc, Chicago) was used for statisticalanalysis with logarithmic transformation of skewed data includingtriglyceride and ACR. Continuous variables were expressed as means±SD orgeometric means×/÷antilog SD where appropriate. Between groupscomparisons were analyzed using Independent Sample T-Test and Analysisof Covariance. The Chi-square test was used to analyze allele andgenotype frequencies and percentage of various diabetes-relatedmetabolic abnormalities and complications. The odds ratio (OR) with 95%confidence intervals (CI) was calculated for the risk of diabeticnephropathy among patients with different number of risk genotypes. A Pvalue of <0.05 (2-tailed) was considered to be significant. Patientswith nephropathy were older with a male predominance compared topatients without nephropathy. After adjustment for age and sex, patientswith nephropathy were more obese with higher body mass index (BMI) andwaist-hip ratio (WHR) as well as higher blood pressure than patientswithout nephropathy. They also had more adverse lipid profile withhigher serum total cholesterol (TC), triglyceride (TG) and lower HDL-Cand were more likely to have sensory neuropathy, retinopathy, peripheralvascular disease and coronary heart disease. Table 1 showed comparisonsof clinical and biochemical parameters between patients with and withoutnephropathy in the 711 Chinese Type 2 diabetes. Data were expressed asMean±SD or a Geometric mean×/÷antilog SD, and were compared byIndependent-Sample T test between patients without and with nephropathy.Percentage of diabetic complications was compared by Chi-square testbetween two study groups.

TABLE 1 Study Groups No Nephropathy Nephropathy Adjusted P (N = 323) (N= 388) P Value Value* Sex (% of Male) 30.0 53.1 <0.001 — Age (years)60.6 ± 10.1 65.1 ± 11.6 <0.001 — Duration of Diabetes (years) 16.0 ±1.6  8.2 ± 5.4 <0.001 — Body mass Index (kg/m²) 23.9 ± 3.2  25.5 ± 4.0 <0.001 <0.001 Waist Height Ratio 0.87 ± 0.06 0.91 ± 0.07 <0.001 <0.001Systolic Blood Pressure (mmHg) 131 ± 18  153 ± 23  <0.001 <0.001Diastolic Blood Pressure (mmHg) 72 ± 10 83 ± 12 <0.001 <0.001 HbA_(1c)(%) 7.6 ± 1.3 8.1 ± 2.1 <0.001 <0.001 Fasting Plasma Glucose (mmol/l)8.3 ± 2.8 9.5 ± 4.4 <0.001 <0.001 Total Cholesterol (mmol/l) 5.2 ± 1.05.9 ± 1.5 <0.001 <0.001 Triglyceride (mmol/l)^(a) 1.1×/÷1.7 1.9×/÷1.9<0.001 <0.001 HDL-C (mmol/l) 1.4 ± 0.4 1.2 ± 0.3 <0.001 <0.001 LDL-C(mmol/l) 3.20 ± 0.84 3.73 ± 1.21 <0.001 <0.001 Plasma Creatinine(μmol/l) 72.4 ± 14.3 152.6 ± 105.4 <0.001 <0.001 Albumin CreatinineRatio (mg/mmol)^(a) 1.1×/÷2.0 128.8×/÷2.9  <0.001 <0.001 Obesity (%)34.5 47.9 <0.001 <0.001 Hypertension (%) 46.7 81.2 <0.001 <0.001Dyslipidaemia (%) 52.0 83.0 <0.001 <0.001 Retinopathy (%) 25.1 54.6<0.001 <0.001 Neuropathy (%) 18.6 43.8 <0.001 <0.001 Peripheral VascularDisease (%) 2.8 13.4 <0.001 <0.001 Ischemic Heart Disease (%) 5.9 12.40.003 0.019 Cerebrovascular Disease (%) 3.7 8.5 0.009 0.098 Use of RASInhibitors (%) 26.9 79.9 <0.001 — *P value after adjustment for age andsex using analysis of covariance.

Table 2 summarized the distribution of these 5 genotypes in patientswith or without nephropathy. Genotype and allele frequencies werecompared by Chi-square test between patients with and withoutnephropathy. Patients with nephropathy had higher frequency of z−2 ofthe 5′-(CA)_(n) (24.1% vs. 18.6%, P=0.01) and T allele of the C-106T(25.8% vs. 21.4%, P=0.05) of ALR2 gene than patients withoutnephropathy. The z−2 allele carriers of ALR2 5′-(CA)_(n) had higher ACRlevel (17.8×/÷12.3 vs. 12.6×/÷12.9 mg/mmol, P=0.062) and percentage ofretinopathy (47.4% vs. 37.4%, P=0.009) than the non z−2 carriers. TheDD/DI genotype carriers of ACE I/D had higher TC level than the IIgenotype carriers (5.7±1.4 vs 5.5±1.3 mmol/l, P=0.047). The obese GGgenotype carriers of TNF-α gene G-308A had higher ACR (22.9×/÷11.5 vs10.7×/÷13.2 mg/mmol, P<0.001) and plasma creatinine level (125±95 vs.108±75 μmol, P=0.005) than non-obese subjects after adjustment for ageand sex.

TABLE 2 Genotype Frequency (%) Allele Frequency (%) Genotypes of No NoGene Nephropathy Nephropathy Alleles of Gene Nephropathy NephropathyPolymorphisms (N = 323) (N = 388) Polymorphisms (N = 646) (N = 776) AGTgene AGT gene M235T M235T TT 70.9 72.9 T 84.4 85.7 TM 26.9 25.5 MM 2.21.5 M 15.6 14.3 ACE Gene I/D ACE Gene I/D II 45.8 46.4 I 66.6 67.3 DI41.5 41.8 DD 12.7 11.9 D 33.4 32.7 TNF-α Gene TNF-α Gene G-308A G-308AGG 80.8 84.0 G 89.9 91.8 GA 18.3 15.5 AA 0.9  0.5 A 10.1 8.2 ALR2 GeneALR2 Gene 5'-(CA)_(n) 5'-(CA)_(n) x/x 67.2 57.7 x 81.4 75.9 x/z − 2 28.536.3 z − 2/z − 2 4.3  5.9* z − 2 18.6 24.1+ ALR2 Gene ALR2 Gene5'-(CA)_(n) 5'-(CA)_(n) y/y 89.9 93.3 y 94.4 96.6 y/z + 6 9.3  6.7 z +6/z + 6 0.9   0** z + 6 5.6 3.4++ ALR2 Gene ALR2 Gene C-106T C-106T CC63.5 56.7 C 78.6 74.2 CT 30.3 35.1 TT 6.2   8.2*** T 21.4 25.8+++ x =any other (CA)n alleles rather than z − 2 allele; y = any other (CA)nalleles rather than z + 6 allele; *P = 0.01, **P = 0.09, ***P = 0.07,when the combined genotype frequencies of ALR2 gene x/z − 2 or z − 2/z −2, y/z + 6 or z + 6/z + 6 and CT/TT were compared between patients withand without nephropathy, respectively; +P = 0.01, ++P = 0.04, +++P =0.05, when the z − 2, z + 6 and T allele frequencies of ALR2 gene werecompared between patients with and without nephropathy, respectively.

Table 3 summarized the distribution of number of genotypes in thispatient cohort. Of the 711 Chinese Tape 2 diabetic patients, 64 (9.0%)had 0 or 1 risk genotype, 176 (24.8%) had 2 risk genotypes, 290 (40.8%)had 3 risk genotypes and 181 (25.5%) had 4 or 5 risk genotypes. Comparedto patients with ≦1 risk genotype, the odds ratio of having nephropathyincreased from 1.4 (95% CI 0.8-2.4, P=0.3) to 1.8 (95% CI 1.1-1.3,P=0.03) and to 2.0 (95% CI 1.1-3.6, P=0.02) in patients with 2, 3 and ≧4risk genotypes, respectively (P=0.006 for trend) (FIG. 1).

TABLE 3 Number of Whole Risk Genotypes No Nephropathy Nephropathy CohortN (%) 0 Risk Genotype 6 2 8 (1.1) 1 Risk Genotype 31 25 56 (7.9) 2 RiskGenotypes 88 88 176 (24.8) 3 Risk Genotypes 125 165 290 (40.8) 4 RiskGenotypes 58 87 145 (20.4) 5 Risk Genotypes 15 21 36 (5.1) Total 323 388711

EXAMPLE 3 Relationship of TNF-α GG Genotypes with Nephropathy

Table 4 indicates the effects of interaction between obesity and G-308Apolymorphism of TNF-α gene on the development of nephropathy in ChineseType 2 diabetic patients, in which “Ref” represent a referent groupusing the non-obese GA/AA carriers. Obese subjects with GG genotype had1.9-fold increased risk (95% CI: 1.1-3.2, P=0.012) for havingnephropathy.

TABLE 4 Risk of Nephropathy Frequency No. (%) P Groups Nephropathy−Nephropathy+ OR 95% CI Value GG−/Obesity− 40 (12.4) 38 (9.8) 1 (Ref.)GG−/Obesity+ 22 (6.8)  24 (6.2) 1.2 0.55-2.38 0.710 GG+/Obesity− 172(53.1)  164 (42.3) 1.0 0.62-1.65 0.970 GG+/Obesity+ 89 (27.6) 162 (41.8)1.9 1.15-3.20 0.012

Table 5 shows the multiple logistic regression analysis to examine therole of interaction of TNF-α gene G-308A with obesity on nephropathyafter adjustment for confounding factors including age, male gender,fasting plasma glucose, hypertension, dyslipidaemia, retinopathy,neuropathy and peripheral vascular disease, the risk for nephropathyincreased by an additional 2.4 fold (95% CI 1.3-4.5, P=0.007) amongstobese patients with GG genotype in 711 Chinese Type 2 diabetic patients.In Table 5, overall percentage correct is 75.2%; dependent variable is:code=1 for nephropathy; independent variables include age, sex (code=1for male), HbA_(1c), FPG, presence of dyslipidaemia, hypertension,retinopathy, neuropathy, peripheral vascular disease, ischemic heartdisease and cerebrovascular disease (code=1), and interaction betweenthe TNF-α gene G-308A polymorphism and obesity is: GG−/Obesity−(code=0), GG−/Obesity+ (code=1), GG+/Obesity−(code=2),GG+/Obesity+(code=3).

TABLE 5 Risk for Having Nephropathy Independent Predictors Odds Ratio95% CI P Value Age (year) 1.02 1.002-1.04  0.033 Male Gender 2.61.78-3.71 <0.001 Fasting Plasma Glucose (mmol/l) 1.1 1.03-1.16 0.002Dyslipidaemia 2.8 1.86-4.18 <0.001 Hypertension 3.2 2.19-4.81 <0.001Retinopathy 2.4 1.62-3.51 <0.001 Neuropathy 2.1 1.36-3.22 0.001Peripheral Vascular Disease 2.8 1.21-6.59 0.017 GG−/Obesity+ vsGG−/Obesity− 1.4 0.57-3.26 0.482 GG+/Obesity− vs GG−/Obesity− 1.20.64-2.15 0.614 GG+/Obesity+ vs GG−/Obesity− 2.4 1.27-4.48 0.007

EXAMPLE 4 Relationship of Ace II/DD Genotype with Nephropathy

In an expanded cohort of 947 Chinese Type 2 diabetic patients with amean follow-up period of 4.0±1.4 years, we examined the impact of ACEII/DD genotype on development of ESRD defined as renal death or events(need for dialysis or plasma creatinine>500 μmol/l or doubling of plasmacreatinine of baseline value>150 μmol/l). Of these 947 patients, 62patients developed renal endpoint.

Table 6 shows the genotype and allele frequencies of ACE I/Dpolymorphism in 947 Chinese Type 2 diabetic patients with and withoutoccurrence of renal endpoint defined as doubling of baseline plasmacreatinine or need for dialysis after a median follow-up period of 4.0years. Genotype and allele frequencies were compared by Chi-square testbetween patients with and without renal endpoints.

From Table 6, the former group had higher DD genotype (19.4% vs. 9.8%,P=0.031) and D allele frequency (41.2% vs. 30.2%, P=0.011) than patientswho did not develop renal endpoint. Kaplan-Meier analysis showed thatthere was significant difference in cumulative renal survival rate amongpatients with II (23 of 460 patients developed renal endpoint), DI (27of 388 patients developed renal endpoint) and DD (12 of 99 patientsdeveloped renal endpoint) genotypes (log-rank P=0.019) (FIG. 2).

TABLE 6 Status of Occurring of Renal Endpoint Non-Occurrence OccurrenceGenotype Frequency (%) II 49.4 37.1 DI 40.8 43.5 DD 9.8 19.4† Total No.of Genotype 885 62 Allele Frequency (%) I 69.8 58.8 D 30.2 41.2‡ TotalNo. of Allele 1771 124 †P = 0.031, ‡P value = 0.011 when the genotypeand allele frequencies were compared between patients with and withoutoccurrence of renal endpoint, respectively.

Table 7 shows the multiple Cox-regression analysis to examine thepredictors for renal endpoint in 947 Chinese Type 2 diabetic patients,in which “a” represents patients who were present of micro- ormacroalbuminuria at baseline; “b” represents DI genotype carrierscompared to II genotype carriers; and “c” represents DD genotypecarriers compared to II genotype carriers. Dependent variables include:Renal death and event (code=1), and independent variables include: age,sex (code=1 for male), duration of diabetes, SBP, DBP, TC, log value ofTG, HDL-C, LDL-C, presence of complications (code=1) includingnephropathy, retinopathy, neuropathy and peripheral vascular disease atbaseline, as well as ACE gene I/D polymorphism (code=1 for DI vs. II,code=2 for DD vs. II).

In multiple Cox-regression analysis, the occurrence of renal endpointremained significantly influenced by I/D polymorphism with a dominantdeleterious effect of the DD genotype (DD vs. II, adjusted hazard ratio3.4, 95% CI 1.6-7.3, P=0.002). Other independent predictors includedlong duration of diabetes, high systolic blood pressure, triglyceride,presence of nephropathy and retinopathy at baseline.

TABLE 7 Independent Hazard Variables (at Baseline) β Coefficient Ratio95% CI P Value Duration of Diabetes 0.050 1.05 1.01-1.09 0.013 (years)Systolic Blood Pressure 0.014 1.01 1.00-1.03 0.012 (mmHg) Log Value of2.063 7.87 2.24-27.7 0.001 Triglyceride (mmol/l) Presence of 3.579 35.8 4.84-265.2 <0.001 Nephropathy^(a) Presence of Retinopathy 0.850 2.341.27-4.30 0.006 DI Genotype Carriers of 0.595 1.81 0.99-3.31 0.053 ACEGene I/D^(b) DD Genotype Carriers of 1.223 3.40 1.59-7.27 0.002 ACE GeneI/D^(c)

EXAMPLE 5 Relationship of ALR2 Genotypes with Nephropathy

In a consecutive cohort of 738 Chinese Type 2 diabetic patients [age55.5±13 years, known disease duration 5.7±5.7 years, means±SD], 21.5%had nephropathy (DN) only, 8% had retinopathy (DR) only, 16.4% had bothnephropathy and retinopathy (DNDR) and 53.1% were free fromcomplications (UC). The CT/TT genotype carriers (N=267) had a higherurinary AER than the CC genotype carriers (N=471) (30.2×/÷7.2 vs.21.9×/÷6.9 μg/min, P=0.03). This difference remained significant(P=0.04) after adjustment for confounding variables including age,duration of diabetes, blood pressure and HbA_(1c).

Since duration of disease is a major determinant for development ofdiabetic microvascular complications (Rogus J. J, Warram J. H, KrolewskiA. S, Genetic studies of late diabetic complications. The overlookedimportance of diabetes duration before complication onset, Diabetes,2002; 51:1655-1662), patients with less than 5 years of diabetes (n=300)were excluded in a subsequent analysis. The remaining patients (n=438)were divided into four subgroups: 159 (36.3%) patients with DN only, 66(15.1%) patients with DR only, 121 (27.6%) patients with both DNDR and92 (21%) patients were uncomplicated (UC). Univariate analysis revealedhigher frequencies of the z−2 allele (25.7% vs. 16.9%, OR 1.7, 95% CI1.0-2.8, P=0.03) and the T allele (26.4% vs. 18.5%, OR 1.6, 95% CI1.0-2.7, P=0.04) in the DNDR group compared with the UC group.

Using age, sex, duration of disease, BP, metabolic indices and the threeALR2 genotypes (z+6 carrying, z−2 carrying and CT/TT) as independentvariables and the UC group as control (code=0), the z−2 carrying (OR2.64, 95% CI 1.02-5.83) and CT/TT genotypes (OR 2.48, 95% CI 1.19-5.19)together with age (OR 1.06, 95% CI 1.02-1.10), BP (OR 1.04, 95% CI1.02-1.06), HbA_(1c) (OR 1.23, 95% CI 1.03-1.46), log TG (OR 20.1, 95%CI 3.73-107.7) and male gender (OR 2.25, 95% CI 1.10-4.61) wereindependent risk factors for prediction of DNDR with 76.9% correct rate.

1. A method for detecting a diabetic subject at risk for developing anephropathy, comprising the step of: determining whether a sample fromthe subject has polymorphic sequences comprising an I/D genotype of anangiotensin converting enzyme (ACE) gene, an M235T genotype of anangiotensionogen (AGT) gene, or an aldose reductase (ALR2) gene 5′-(CA)repeats, or a complement thereof, wherein the presence of thepolymorphic sequence indicates the subject is at risk for developing thenephropathy.
 2. The method of claim 2, wherein the sample is blood. 3.The method of claim 2, which further comprises the step of amplifyingthe ACE and ALR2 genes.
 4. The method of claim 3, wherein primers usedfor amplifying step is performed with primers having SEQ ID NO. 1 andSEQ ID NO. 2 for an I/D genotype of the ACE gene, SEQ ID NO. 3 and SEQID NO. 4 for an M235T genotype of the AGT gene, and SEQ ID NO. 7 and SEQID NO. 8 for a (z−2) genotype of the ALR2 gene.
 5. The method of claim1, wherein the subject is at risk for developing Type 2 diabetes.
 6. Themethod of claim 1, wherein the I/D genotype comprises a DD genotype. 7.The method of claim 1, wherein the polymorphic sequences furthercomprise a (z−2) genotype of a G-308A genotype of a tumor necrosisfactor alpha (TNF-α) gene, or an C106T genotype of an ALR2 gene in thepromoter region.
 8. The method of claim 7, wherein the primers used foramplifying further comprise SEQ ID NO. 5 and SEQ ID NO. 6 for a G-308Agenotype of the TNF-α gene, and SEQ ID NO. 9 and SEQ ID NO. 10 for aC106T genotype of the ALR2 gene in the promoter region.
 9. The method ofclaim 7, wherein the G-308A genotype comprises a GG genotype.